We are developing HIV pDNA based vaccine strategies both as preventive and therapeutic strategies. Attractive features of the pDNA platform lie in its simplicity, versatility, stability, with repeated administration without vector immunity, being a non-replicating vaccine and not association with adverse effects. There is a licensed pDNA vaccine against dog melanoma. Several pDNA vaccines are currently in clinical trials including against cancer. We are testing immunogenicity of SIV and HIV pDNA vaccines in mice and selected pDNA candidates are tested in the macaque model. Some of our successful candidates are now moving to clinical trials. This work is based on our previous recognition that RNA elements (called INS) present within the gag/pol and env coding regions of HIV are responsible for nuclear retention and instability of the transcripts in the absence of Rev, and that these elements can be eliminated by changing the nucleotide composition of the transcripts (RNA optimization; also referred to as codon optimization) without affecting the amino acid sequence. The immunogenicity of the antigens is further improved by modifying the trafficking of the antigens which is derived from our in-depth analysis of the molecular biology of the proteins. Because intramuscular injection of pDNA induces relatively low immune responses in macaques and humans, we are testing additional delivery methods, including in vivo electroporation, liposomes. We reported that electroporation dramatically increased the efficiency of DNA delivery in naive macaques, leading to greatly augmented antigen expression and resulting in the induction of highest levels of T cell responses. Our pDNA vaccine includes the cytokine IL-12 pDNA as adjuvant, increasing magnitude and quality of the responses. Importantly, we reported the dissemination of the pDNA vaccine induced T cell responses to mucosal sites including rectal and vaginal mucosa, the portal of entry of HIV. We also found that pDNA induced immune responses show extraordinary longevity in vaccinated macaques detectable for several years after the last vaccination. pDNA vaccination elicits moderate humoral immune responses in macaques. We showed that a protein boost can induce higher levels of Ab. We found that co-delivery of pDNA+protein either unadjuvanted or adjuvanted in the same muscle at the same time increased Ab production and mucosal dissemination. DNA+Protein co-immunization is superior to vaccination with either of the two individual components in eliciting humoral immune responses. Using pDNA-only vaccination, we found that our optimized DNA vaccine vectors are able to induce potent immune responses able to protect from high viremia. Combination of pDNA+Protein induces potent humoral responses able to significantly delay or prevent virus acquisition and improvement in virological control of the highly pathogenic SIV challenge. As in the RV144 trial, our vaccine regime induces responses to the V2 region that have been associated with delayed virus acquisition in humans. We are now exploring novel Env vaccination protocols and novel protein adjuvants to improve vaccine efficacy. In a collaborative effort, we are testing the concept of combining vaccine against rubella and HIV. We focused on Gag as antigen, because Gag-specific T cell responses were reported to correlate with control of viremia in infected individuals and such responses are expected to reduce viremia in both preventive and therapeutic vaccination protocols. Vaccine regimens including rubella vector and SIV gag DNA in different prime-boost combinations resulted in robust long-lasting cellular responses with significant increase of cellular responses upon boost. Rubella vectors provide a potent platform for inducing HIV-specific immunity that can be combined with DNA in a prime-boost regimen to elicit durable cellular immunity. We continue to build on this vaccine platform. An ideal HIV vaccine should provide protection against all HIV-1 variants. Thus, an important aspect of HIV vaccine development is the selection of the immunogens, which has to take into consideration the diversity of the different HIV clades and the identification of the critical epitopes able to induce relevant immune responses, avoiding potential immunodominant decoy epitopes. Indeed, we reported a potent impact of Env on the induction of Gag cellular responses. To address these problems, we are exploring approaches to maximize immunological strength and breadth using mosaic and consensus molecules as well as focusing on highly conserved regions of HIV to induce immune responses to nearly invariable proteome segments, essential for the function of the virus, while excluding responses to variable and potentially immunodominant decoy epitopes. Using the latter, we have developed two distinct approaches focusing on conserved regions of HIV. (1) The HTI immunogen contains highly conserved regions of gag, pol, vif, and nef and includes optimally defined CD4+ and CD8+ T-cell epitopes restricted by a wide range of HLA class I and II molecules and covers viral sites where mutations led to a dramatic reduction in viral replicative fitness. In mice and macaques, immunization with DNA.HTI induced broad and balanced T-cell responses that were increased by a booster vaccination using MVA.HTI. These data demonstrate the immunogenicity of a novel T cell vaccine that induced broadly balanced responses to vulnerable sites of HIV while avoiding the induction of responses to potential decoy targets that may divert effective T-cell responses towards variable and less protective viral determinants. The DNA.HTI prime-HTI.MVA boost vaccine strategy is currently developed in Europe for a clinical trial. (2) We developed another prototype vaccine targeting highly conserved regions within the p24gag (p24CE DNA vaccine). Immunogens were engineered encoding conserved elements (CE) of HIV-1 selected on the basis of stringent conservation, functional importance, broad HLA-coverage, and association with viral control. In proof-of-concept studies in mice and macaques, we demonstrated that immunization with CE pDNA elicits robust cellular and humoral immune responses against CE, which cannot be achieved by p55gag DNA vaccination. This vaccine induces potent cytotoxic T cells responses targeting the Achilles' heel of the virus and induces robust immune responses to subdominant epitopes. Importantly, we demonstrated that priming with CE pDNA and boosting with p55gag pDNA greatly augment the CE-specific responses and that inclusion of the p24CE+gag pDNA in the boost maximizes both magnitude and breadth. We identified a novel and effective strategy to maximize responses against Gag and provide a novel strategy to shift the immunodominance hierarchy and to induce robust immune responses to subdominant epitopes. Translation of this novel vaccine concept is currently being pursued in an HVTN/DAIDS-supported clinical trial with the aim to test whether our p24CE pDNA vaccine concept elicits superior breath and magnitude of Gag responses compared to the optimized immunogen comprising the complete p55Gag protein. cGMP preparations of our pDNA molecules have been generated and tested for potency and immunogenicity. This vaccine includes IL-12 pDNA as molecular adjuvant and in vivo electroporation as pDNA delivery method, two vaccine components our research had shown to be of outmost importance to induce potent T cell responses in macaques. Thus, this trial will combine several of milestones (pDNA expression vectors, adjuvants and delivery) we have achieved over many years in vaccine development.